Treating male infertility secondary to sperm oxidative stress

ABSTRACT

A profertility composition for administration to infertile men with sperm oxidative stress is disclosed. The composition comprises a unique combination of a pharmaceutically acceptable vitamin E, vitamin D, vitamin C, selenium, zinc, folate, lycopene, at least one carnitine source, and optionally Coenzyme Q10. Also disclosed are methods of treating male infertility.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No. 15/953,939, filed on Apr. 16, 2018, which is a Continuation of U.S. patent application Ser. No. 13/749,177, filed on Jan. 24, 2013, now U.S. Pat. No. 9,943,543, which is a Continuation of U.S. patent application Ser. No. 12/771,928, filed on Apr. 30, 2010, now U.S. Pat. No. 8,377,454, which claims priority to U.S. Provisional Patent Application No. 61/174,732 filed on May 1, 2009, the contents of each of which is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to methods, compositions and fertility kits used to enhance the natural fertility potential of men. The present invention utilizes naturally occurring antioxidants to provide a prophylactic treatment of sperm oxidative stress. Additionally, the novel products described herein further include vitamins and minerals for improving semen parameters enhancing the overall health of sperm thus increasing fertilization rates, number of viable embryos, pregnancy rates, and reducing miscarriages.

BACKGROUND

Male infertility has been on the rise for several decades and is now considered a major health problem affecting around one in twelve of the men in USA. If the trends observed over the last century continues, some infertility experts predict that by the middle of this century, most men will be advised to freeze their sperm at a young age to ensure adequate sperm quality.

A successful fertilization and subsequent embryo development requires properly matured sperm chromatin with minimal damage to its DNA integrity. Equally important, is the integrity of sperm membrane lipid structure essential for sperm motility and sperm-oocyte fusion. At least one of the causes of sperm DNA damage and membrane peroxidation is attributed to excess Reactive Molecular Species (“RMS”), most importantly Reactive Oxygen Species (“ROS”), leading to a condition commonly referred to as sperm oxidative stress. In general, the term “Oxidative Stress” refers to a condition that occurs as a result of excessive generation of ROS and/or a diminished capacity of ROS scavenging endogenous antioxidants. Accordingly, the excess concentration of ROS overwhelms the local environmental antioxidant defense mechanism of the spermatozoa potentially leading to the formation of sperm cells with lower or no fertilizing potential.

Under normal conditions, cells utilize an array of enzymatic and non-enzymatic antioxidants as internal defense to scavenge and neutralize excess levels of ROS. It is believed that oxidative stress develops either because of an overabundance of ROS from environmental or pathological stressors or a lack of antioxidant capacity to neutralize excess concentrations of ROS. Excess ROS within semen and the sperm cell has been described to cause degradation of the cellular components such as membrane and DNA, hence, the sperms injured by such oxidative species have diminished or no fertility potential.

To date several studies have reported that levels of ROS within semen can be reduced by augmenting the scavenging capacity of seminal plasma using oral antioxidant supplementation. In fact, the benefits of oral antioxidant therapy has been described in numerous articles. By the way of reference, such articles include Comhaire et al, The Effects of Combined Conventional Treatment, Oral Antioxidants and Essential Fatty Acids on Sperm Biology in Subfertile Men, Prostaglandins Leuko Essent Fatty Acids, 63, 159-165 (2000); Lenzi et al, Placebo-Controlled, Double-blind, Cross-over Trial of Gluthathione therapy in male infertility, Hum Reprod, 8, 1657-1662 (1993); Greco et al, Reduction of the Incidence of Sperm DNA Fragmentation by Oral Antioxidant Treatment. J Androl, 17, 276-287 (2005); Tremellen et al, A Randomised Control Trial Examining the Effect of an Antioxidant (Menevit) on Pregnancy Outcome During IVF-ICSI Treatment, Australian and New Zealand Journal of Obstetrics and Gynecology 47, 216-221 (2007); and Agarwal et al, Oxidative stress, DNA Damage and Apoptosis in Male Infertility, a Clinical Approach, BJU Int, 95, 503-507 (2005), the teachings of which are incorporated herein in their entirety.

However, there is no product in the market today that has the instantly disclosed combination of antioxidants, minerals and vitamins. Moreover, the prior art does not teach the steps described herein to enhance the natural fertility process. At least one object of the present invention is to address such a need in the field.

Excessive concentration of ROS can cause extensive sperm DNA damage and membrane peroxidation. In general ROS includes reactive moieties such as hydroxyl radicals (OH), ionic species such as superoxide anions (02), and neutral but highly reactive oxidizing molecules such hydrogen peroxides (H₂O₂). In semen, such species are predominantly generated by leukocytes or morphologically abnormal sperms. It has been described in the art that ROS plays both physiological and pathological roles in maturation of sperm cells. For example, at normal baseline ROS concentrations, sperms are properly matured as ROS modulates the regulation of the normal sperm function in various cascades including but not limited to sperm-oocyte fusion, acrosome reaction and sperm capacitation.

On the other hand, excess ROS concentrations result in direct insult on cellular components such as lipids, proteins and DNA, thus interfering with normal cell function. Traditional semen analysis methodologies fail to detect DNA damage caused by ROS. In fact, in many cases infertile men who undergo a clinical workup show normal sperm parameters, including sperm count motility and morphology. However, when compared with healthy fertile men, at least a subpopulation of these infertile men, show higher concentrations of oxidants in their seminal fluid which may be coupled with significantly lower concentrations of antioxidant as compared to healthy fertile men.

At the genomic level, excess concentrations of ROS modifies sperm DNA structure in a number of ways. For example, ROS can induce chromatin cross-linking, DNA base oxidation and cause high frequencies of single and double DNA strand breaks. Even though, sperms with damaged DNA may still fertilize the oocyte, the degree of DNA damage can have profound implications in normality of embryonic development resulting in higher miscarriage rates, birth defects or the long-term health of the progeny.

Current medical approach to facilitate embryonic development and pregnancy uses invasive techniques collectively referred to as Assisted Reproductive Techniques (“ART”), including Intrauterine Insemination (“IUI”), In vitro Fertilization (“IVF”) and Intracytoplasmic Sperm Injection (“ICSI”). However, such techniques generally have an average rate of success of about 30%. At least one explanation for such poor outcome is the fact that the semen and the sperm analysis techniques employed during ART cannot differentiate sperms with DNA damage from the normal ones.

Moreover, ART modes of fertilization have been linked with birth defects and incidences of childhood cancer, e.x. acute leukemia and lymphoma. Nevertheless, ART remains the only viable option for infertile couples. Another shortcoming of the ART is the cost. Couples on average undergo several IVF attempts to achieve a pregnancy, therefore undertaking huge expenses. Additionally, the risk of failure often causes intense emotional trauma. Moreover, ART cannot and does not address the damage done by ROS to sperm and ultimately to the fertilized egg. Therefore, there is a need in the art to address the impact of Sperm Oxidative Stress (“SOS”) among infertile couples. Currently, there are no effective and generally accepted pharmacotherapy that can combat the problem of male infertility associated with SOS. Thus, there is a need for convenient, inexpensive fertility methodologies and products to aid and improve couple's fertility potential. There is also a need for a kit that will provide couples in need with all products, tools and material required to facilitate a comprehensive plan for achieving a successful pregnancy.

SUMMARY OF THE DISCLOSURE

The present invention fills the foregoing need by lowering and prophylactically treating SOS by boosting the non-enzymatic antioxidant defense mechanism. The invention comprises unique combinations of several natural antioxidants, vitamins and minerals clinically tested for efficacy to improve sperm health and quality. The invention is therefore intended to be a prophylactic treatment to improve the overall health of sperms thus improving the sperm-oocyte fertilization, reducing miscarriage rates and improving pregnancy rates.

This invention provides methods of using combinations of naturally occurring antioxidant molecules for treating infertility in men predominantly suffering from SOS alone or in combination with aromatase inhibitors, suitable anti-inflammatory, and/or antibiotics.

At least one aspect of the present invention is directed to a method of increasing the rate of pregnancy in a female subject by administering to the male partner an effective amount of a combination of antioxidants, vitamins and minerals, for at least a period of up to about three (3) months prior to any fertilization attempts, wherein said combination is substantially free of vitamin A, vitamin K and/or garlic. In at least one embodiment of this aspect of the invention, the combination includes at least one source of vitamin C, at least one source of vitamin E, at least one source of carnitine, lycopene and at least one source of folic acid.

In a more preferred embodiment of this aspect of the invention, the combination includes at least one source of vitamin C, at least one source of vitamin E, at least one source of carnitine, lycopene and at least one source of folic acid, at least one mineral which is selenium, zinc, magnesium or combinations thereof, wherein the composition is free of any traces of vitamin A, vitamin K and/or garlic. The composition can also contain vitamin D and/or coenzyme Q10 (CoQ10). Preferably the vitamin E is natural vitamin E.

In another aspect of the present invention, a novel method of reducing ROS damage to sperm DNA by at least 2.5% as compared with placebo, including the steps of administering to a male subject for at least a period of up to about three (3) months an effective amount of the combination of antioxidants, vitamins and minerals disclosed herein, wherein the combination is free of vitamin A, vitamin K and/or garlic.

In at least one embodiment of this aspect of the invention, the method of inhibiting ROS damage to sperm DNA cause a reduction in a measurement of DNA fragmentation secondary to SOS by at least 1%, 2.5%, 5%, 8%, 10%, 12.5%, 15%, 20%, 30% and preferably higher when compared to a placebo treatment. In another embodiment, the sperm DNA fragmentation is assessed by such technique including but not limited to Sperm Chromatin Dispersion (“SCD”) test, Sperm Chromatin Structure Assay (“SCSA”), DNA Breakage Detection-Fluorescence In Situ Hybridization (“DBD-FISH”) test, Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nic-End Labelling (“TUNEL”) assay and 8-hydroxy-deoxyguanosine Assay (“8-OH-dG Assay”).

Another aspect of the present invention provides for a method of treating an infertile male subject with sperm oxidative stress including the steps of (a) ascertaining the degree of DNA damage in said male subject, as measured by SCSA, TUNEL, SCD, DBD-FISH, or 8-OH-dG Assay, (b) initiating a course of therapy by administering an effective amount of a combination of antioxidants, vitamins and minerals and free of vitamin A, vitamin K and/or garlic for at least a period of three (3) months, (c) re-ascertaining the degree of DNA damage in said male subject post the therapy of the step (b), and (d) fertilizing the egg of the female partner with said male subject's sperm after completion of the treatment course of step (b), wherein the degree of the DNA damage measurement is reduced or otherwise improved, as compared to placebo or said subject's own pretreatment values by at least 1%, 2.5%, 5%, 8%, 10%, 12.5%, 15%, 20%, 30%, and preferably higher.

Another aspect of the present invention provides for a method of treating an infertile couple including the steps of (a) determining the couples susceptibility to oxidative stress, (b) initiating the combination regimen disclosed herein for the male partner wherein the male partner receives an effective amount of a combination of antioxidants, vitamins and minerals and free of vitamin A, vitamin K and/or garlic for at least a period of three (3) months. In another aspect of the present invention, the female partner may independently or simultaneously with the male partner receive the antioxidant therapy optionally including an alpha-lipoic acid regimen.

The present invention also provides a method of reducing the concentration of ROS generated by leukocytes in the reproductive tract and/or semen of a male subject, the method including the steps of administering to the male subject (a) an effective amount of the antioxidant, vitamin and mineral combination for at least a period of three (3) months and (b) an effective amount of an aromatase inhibitor agent or an appropriate antibiotic.

The present invention also provides a method of improving sperm function in a male subject, the method including the steps of administering to the male subject an effective amount of the combination described herein for at least a period of three (3) months.

The present invention also provides for a method of improving quality of an embryo produced by fertilization of an oocyte by a sperm from a male subject, wherein the male subject has completed a three (3) month course of antioxidant regimen comprising receiving a combination of at least one source of vitamin C, at least one source of vitamin E, at least one source of carnitine, and at least one source of folic acid, at least one mineral which is selenium, zinc, magnesium or combinations thereof, wherein the regimen is free of any traces of vitamin A, vitamin K and/or garlic. The combination can also include lycopene and/or vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E.

In at least one embodiment of this aspect of the invention, methods of prophylactically reducing the risk of forming a nonviable zygote comprising administering to a male subject in need thereof, a composition comprising an effective amount of a combination of at least one source of vitamin C, at least one source of vitamin E, at least one source of carnitine, and at least one source of folic acid, at least one mineral which is selenium, zinc, magnesium or combinations thereof, wherein the regimen is free of any traces of vitamin A, vitamin K and/or garlic, and optionally an effective dose of an aromatase inhibitor or an antibiotic. The combination can also include lycopene and/or vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E.

The present invention also provides a composition comprising an effective amount of at least one source of vitamin C, an effective amount of at least one source of vitamin E, an effective amount of at least two sources of carnitine, and an effective amount of at least one source of folic acid, at least one mineral which is selenium, zinc, magnesium or combinations thereof wherein the composition is substantially free of any traces of vitamin A, vitamin K and/or garlic. The combination can also include lycopene and/or vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E. In a preferred embodiment of this aspect of the invention, the composition only contains an effective amount of at least a source of vitamin C, an effective amount of at least one source of vitamin E, an effective amount of L-carnitine, propionyl-L-carnitine or acetyl-L-carnitine or combinations thereof, and an effective amount of at least one source of folic acid, an effective amount of lycopene, and an effective amount of at least one mineral which is selenium, zinc, magnesium or combinations thereof, wherein the composition is free of any traces of vitamin A, vitamin K and/or garlic. The combination can also include vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E.

In a preferred embodiment of this aspect of the present invention, a composition is disclosed that contains vitamin E, vitamin C or a suitable salt thereof, folic acid, lycopene, a carnitine source, selenium, and zinc in pharmaceutically acceptable forms wherein the formulation is substantially free of vitamin A and vitamin K and/or garlic or any extracts thereof. The composition can also include vitamin D and/or coenzyme Q10. Preferably the vitamin E is natural vitamin E.

In a more preferred embodiment of this aspect of the invention the composition is composed of vitamin E in about 100-800 IU; vitamin C about 50-800 mg, or an equivalent salt thereof selenium in about 25-100 μg; zinc in about 10-35 mg; folic acid in about 0.25-0.75 mg; lycopene about 3-12 mg; and a carnitine source in about 250-4000 mg, wherein the formulation is substantially free of vitamin A and vitamin K and/or garlic or any extracts thereof. The combination can also include vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E. In yet another embodiment of the present invention, the carnitine source is a prodrug for carnitine.

In the preferred embodiment of the present invention the composition includes vitamin E in about 200 IU; vitamin C in about 90 or 200 mg, selenium in about 55 μg; zinc in about 15 mg; folic acid in about 0.5 mg; lycopene about 5 mg; and acetyl-L-carnitine in about 1000 mg. The combination can also include lycopene and/or vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E. In yet another embodiment, the composition includes vitamin E in about 200 IU; vitamin C in about 200 mg, selenium in about 55 μg; zinc in about 15 mg; folic acid in about 0.5 mg; lycopene about 5 mg; and propionyl-L-carnitine in about 500 mg, and acetyl-L-carnitine in about 1000 mg. The combination can also include lycopene and/or vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E.

Another aspect of the present invention is directed to the disclosed antioxidant containing compositions, wherein the compositions are in the form of a tablet, a capsule, a softgel, a powder mixture, a paste, a suspension, a solution, an elixir, a topical or an oral patch, or a parenteral preparation. In at least on embodiment of this aspect of the invention, the composition contains sustain or extended release components, immediate release components, microparticles, nanoparticles and suitable excipients to enhance the bioavailability of the product as compared to their respective conventional preparations. In the preferred embodiment of this aspect of the invention, the antioxidant containing composition can be administered orally, via an inhaler, or via the parenteral route.

In another aspect of the present invention, pregnancy kits are provided comprising a set of pregnancy tests, articles for storage of specimens, and compositions containing vitamin E in about 200 IU; vitamin C in about 90 or 200 mg, selenium in about 55 μg; zinc in about 15 mg; folic acid in about 0.5 mg; lycopene about 5 mg; and acetyl L-carnitine in about 500 mg and/or compositions containing vitamin E in about 200 IU; vitamin C in about 90 or 200 mg, selenium in about 55 μg; zinc in about 15 mg; folic acid in about 0.5 mg; lycopene about 5 mg; and propionyl-L-carnitine in about 500 mg. The combination can also include lycopene and/or vitamin D and/or CoQ10. Preferably the vitamin E is natural vitamin E.

In at least another aspect of the present invention, pregnancy kits can comprise additional adjuvant agents such as aromatase inhibitors, glutathiones, or Coenzyme Q10, each of which may be administered separately or in combination with the antioxidant, mineral and vitamin combinations instantly disclosed.

The present invention also provides a method of isolating sperm and/or measuring DNA fragmentation from a male subject, the method including the steps of (a) administering to the male subject an effective amount of at least one source of vitamin C, an effective amount of at least one source of vitamin E, an effective amount of at least one source of carnitine, and an effective amount of at least one source of folic acid, an effective amount of lycopene, at least one mineral which is selenium, zinc, magnesium or combinations thereof wherein the composition is substantially free of any traces of vitamin A, vitamin K and/or garlic and (b) measuring the subject's sperm's DNA fragmentation index.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents % mouse sperm DNA damage upon treatment of mice with 3 different xenobiotics that cause oxidative stress (efavirenz, acetaminophen (ACE), and paroxetine) followed by F1/6, F1/3 and FMAX.

FIG. 2 presents % mouse sperm chromatin defect upon treatment of mice with 3 different xenobiotics that cause oxidative stress (efavirenz, acetaminophen, and paroxetine) followed by F1/6, F1/3 and FMAX.

FIG. 3 presents pregnancy rates and litter data produced from male mice treated with efavirenz, followed by F1/6, F1/3 and FMAX.

FIG. 4 presents pregnancy rates and litter data produced from male mice treated with acetaminophen (ACE), followed by F1/6, F1/3 and FMAX.

FIG. 5 presents pregnancy rates and litter data produced from male mice treated with paroxetine, followed by F1/6, F1/3 and FMAX.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs and shall be understood to have the meanings described below. All publications and patents referred to herein are incorporated by reference in their entirety. Unless otherwise specified, a reference to a particular compound includes all ionic, salt, solvate (e.g., hydrate), protected forms, prodrugs, and other stereoisomers thereof, isomeric forms, including racemic and other mixtures thereof. The invention provides the first products to put these components together to act in a synergistic manner in men's formulations. The invention also provides for the very first time the unexpected reduction of the DNA sperm fragmentation by the combination described herein:

The term “aromatase inhibitors” is used herein to refer to reversible or irreversible aromatase inhibitors including but not limited to natural flavanoids or synthetic products such products as anastrozole and exemestane (which is sold as Aromasin and is chemically described as 6-methylenandrosta-1,4-diene-3,17-dione), analogues or derivatives thereof such as those described in U.S. Pat. Nos. 4,808,616, 4,876,045 and 4,904,650, the teachings of which are incorporated herein in their entirety.

As used herein, “about” will mean up to plus or minus 5% of the particular term.

As used herein, “consisting essentially of” refers to excluding other active ingredients but including excipients.

The term “carnitine,” except as individually recited, includes all pharmaceutically acceptable forms of carnitine including but not limited to L-carnintine or a prodrug thereof, acetyl-L-carnitine and propionyl-L-carnitine in form of HCl salt, in general alkanoyl L-carnitines such as isovaleryl-L-carnitine, hexanoyl-L-carnitine, octanoyl-L-carnitine, myristoyl-L-carnitine, palmitoyl-L-carnitine, stearoyl-L-carnitine.

By “Fertilization Attempt” it is meant any process that facilitates fusion of the sperm and egg. Such term includes natural as well as medical procedures that facilitate embryonic development and pregnancy including but not limited to IUI, IVF and ICSI.

The term “folic acid or folate” (sometimes known as vitamin B9) is meant to include the synthetic form of the vitamin, in hydrate or salt forms with metal ions such as sodium, zinc etc.

The term “lycopene” is meant the red pigment in tomatoes in its pharmaceutically acceptable organic or inorganic form, which contains a C40 open-chain hydrocarbon carotenoid with any of its 11 conjugated double bonds being available for the formation of cis and trans-geometrical isomers.

By the term “Reactive Molecular Species (“RMS”) it is meant to include Reactive Oxygen Species (“ROS”), Reactive Nitrogen Species (“RNS”) and other reactive heteroatom species that can incapacitate sperm ability to fertilize an egg, preferably ROS as the most evaluated reactive moieties.

The term “selenium” is meant to include elemental, all inorganic and organic forms of selenium such as sodium selenite and sodium selenate, selenomethionine, L-selenomethionine, selenocysteine, methyselenocysteine, methylselenol, amino acid chelates, yeast, and kelp bound selenium.

By the term “substantially free” it is meant that the final product is free of any traces of the identified ingredient to the extent that it could be detected by general quantitative assays known in the art for detection of such ingredient in the final product, or if detected it is in residual content of the ingredient and in principle only attributable to impurities.

The term “treatment” or “therapy” as used herein in the context of treating a condition to the extent that a positive clinical benefit is observed. Thus, a course of therapy includes prophylactic treatment, and further pertains generally to the therapy of a human subject. For example, the treatment of sperm oxidative stress and/or male infertility associated with sperm oxidative stress includes a reduction in the rate of production, rate of proliferation or the concentration of ROS in sperm cells, which can result in a halt in the rate of infertility.

Treatment includes combination treatments and therapies, in which two or more treatments or therapies modalities are combined, for example, sequentially or simultaneously. Examples of treatments and therapies include, but are not limited to, antioxidant combination therapy with or without the use of anti-inflammatory agents, surgery, employing ART and/or other drug treatments generally known to improve rate of pregnancy.

The term “therapeutically-effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage form comprising an antioxidant, which is effective for producing the desired therapeutic effect, commensurate with a reasonable benefit/risk ratio to avoid excess concentration of such drugs to the extent that it would neutralize its expected therapeutic effects.

The term “vitamin C and/or its derivatives,” except as individually recited, is meant to include L-ascorbic acid, a mineral ascorbate or a multi-mineral ascorbate, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, molybdenum ascorbate, chromium ascorbate, vitamin C Complex, Ascorbyl palmitate.

The term “vitamin E and/or its derivatives” is meant to include all variations of vitamin E, including but not limited the eight antioxidant isoforms commonly known as vitamin E: four tocopherols (alpha-, beta-, gamma-, and delta-) and four tocotrienols (alpha-, beta-, gamma-, and delta-). The naturally occurring mixture of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol is referred to herein as “natural vitamin E”. Natural vitamin E does not include acetates or other esters.

The term “zinc” is meant to include all variations of zinc including but not limited to elemental zinc, zinc sulphate, zinc picolinate, zinc citrate, zinc acetate, zinc glycerate, and zinc monomethionine.

The invention provides a combination therapy for improving fertility in mammals particularly among those suffering from excess oxidative stress at their reproductive cell levels. At least one aspect of the present invention provides treating infertility among any mammal including but not limited to land and aquatic animals such as primates, horses, donkeys, sheep, cats, dogs, pigs etc. In a more preferred embodiment, the mammal is a male human, horse or cow and in the most preferred embodiment the mammal is human.

In another aspect of the present invention the presently disclosed treatment reduces male infertility associated with sperm oxidative stress. The invention provides a scientifically validated nutritional blend for mammalian male to reduce sperm oxidative stress and infertility associated with such condition and further improve the sperm genomic integrity. The present combination of vitamins, minerals and amino acids reduces overall fragmentation of sperm cells and further secure higher rate of pregnancy among mammalian couples whose male counterpart suffers from infertility.

Those of ordinary skill in the art understand that sperm is highly susceptible to free radical or oxidative damage from environmental toxicants and natural aging. In at least one embodiment of the present invention vitamins C and E, zinc, and selenium, all of which are potent antioxidants that help improve sperm counts and quality, are used to neutralize the 126168692.3 formation of excess ROS and maintain normal DNA makeup. Zinc and B vitamins (B6, B12 and folate) are critical nutrients in male reproductive systems for hormone metabolism, sperm formation and motility. However, synergistic combination of such ingredients for purposes of treating male infertility secondary to SOS has not been clinically proven. At least one aspect of the present invention, describes a profertility combination of such ingredients for treating male infertility associated with SOS.

In another aspect, the profertility combination of the present invention employs a source of carnitine. Such source includes L-carnitine, acetyl-L-carnitine or propionyl-L-carnitine and all pharmaceutically acceptable salts thereof. The preferred embodiment of this aspect of the invention employs propionyl-L-carnitine, hydrochloride, fumarate or tartrate salts thereof.

Another aspect of the present invention provides for a component that would have a synergistic action of the combinations. The distinct combinations are useful for couples who have failed to produce a healthy fertilized oocyte during at least a period ranging from three (3) to six (6) months prior to any fertilization attempts. Such couples generally include male subjects exposed to occupational toxic agents such as styrene, as well as those who may be subject to environmental, chemical, radiation, and heat exposure.

In one embodiment, the male human subject of the present invention is selected from the group consisting of a subject with increased levels of sperm DNA oxidation, reduced fertility of unknown origin; a subject having undergone vasectomy reversal; a subject with a reproductive tract infection such as epididymitis; and a subject having a varicocele.

Previous human studies assessing male subjects with increased levels of malondialdehyde or other biochemical markers of oxidative stress; a smoker; a subject with reduced fertility, including reduced fertility due to poor sperm motility have all failed to substantiate the use of other antioxidants' combinations in treating DNA fragmentation secondary to sperm oxidative stress. See Tremellen supra.

It has unexpectedly been discovered that the instantly claimed blend of antioxidants, when administered in an appropriate dose range, drastically reduces sperm DNA fragmentation from the pretreatment baseline and increases rate of pregnancy.

Methods are known in the art for assessing the extent of free radical damage to sperm. For example, the thiobarbituric acid reactive substances (“TBARs”) assay (which involves the measurement of malondialdehyde, a marker of sperm membrane oxidation) or LPO-856 spectrophotometric assay may be used. These methods are described for example in Gomez et al International Journal of Andrology 21(2), 81-96 (1998). Other methods include measurement of DNA Fragmentation Index (“DFI”) using SCSA, SCD test, DBD-FISH test, and 8-OH-dG Assay.

In one embodiment, the administration of the present combination therapy to the subject results in a reduction in free radical damage to sperm's DNA of at least 2.5%, 5%, 8%, 10%, 12.5%, 15%, preferably 20% and most preferably 30%, when measured by a suitable sperm parameter analysis, preferably evaluated or expressed as DFI. In at least another embodiment, the administration of the present combination therapy to the male subjects results in a significant improvement in the degree of DNA damage secondary to SOS, when compared to the subject's own pretreatment values, by at least 1, 2.5%, 5%, 8%, 10%, 12.5%, 15%, 20%, 30%, and preferably 50%, wherein such improvement advances the pregnancy of a female partner. In a preferred embodiment, the administration of the antioxidant composition to a male subject results in a reduction in pretreatment baseline levels of the subject's DFI measurement by at least an absolute 2.5% (i.e. the actual difference in DFI measurement before and after the combination therapy).

The antioxidant agent in the various embodiments of the present invention may be one or more individual antioxidant(s). In this regard, an antioxidant is a molecule that can directly or indirectly reduce the damaging effects of oxygen and/or free-radicals in cells, and includes molecules that react with oxygen, or molecules that may protect against, and/or react with, a free radical.

In one embodiment, the antioxidant agent is selected from one or more of the group consisting of a vitamin C; vitamin D; vitamin E; β-carotenoid, including lycopene (a carotenoid derived from the tomato), lutein; a folic acid or derivative, selenium; zinc; L-carnitine, a prodrug thereof such as propionyl-L-carnitine; acety-L-carnitine; N-acetylcysteine; glutathione; gluthathionine; pyruvate; Coenzyme Q10 (CoQ10), astaxanthin and hypotaurine; alpha-lipoic acid or a salt (if applicable), or a pharmaceutically acceptable derivative of any of the aforementioned agents. Other antioxidants are generally as described in Agarwal et al, Role of Antioxidants in Treatment of Male Infertility: an Overview of the Literature, Reproductive Biomedicine Online, 8(6), 616-62 (2004). Compounds such as gluthathione, astaxanthin or Coenzyme Q10 may be administered parenterally to enhance the ultimate clinical antioxidation.

The effective amount of the one or more antioxidant agents in the various embodiments of the present invention may be limited, and it is important that the antioxidant has the desired or therapeutic effect, which will depend upon the particular antioxidant(s) administered. In a preferred embodiment, the present invention contains tablets, capsules, softgels, or some combination thereof having:

1) natural vitamin E or d-alpha-tocopheryl acetate in a range of about 50 to about 1500 I.U., with a usual range of about 200 to about 1200 I.U., and typically about 100 to about 800 I.U. or about 200 to about 800 I.U., alternatively about 10 to about 500 mg of natural vitamin E, or about 25 to about 400 mg, or about 38 to about 235 mg, or about 40 to about 200 mg, or about 100 to about 175 mg, or about 112 to about 188 mg, preferably about 150 mg; alternatively the natural vitamin E can be present in about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 270, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg of natural vitamin E;

2) vitamin D in ranges of about 400 to about 3200 I.U., or about 600 to about 3000 I.U., or about 800 to about 2400 I.U., or about 1000 to about 2000 I.U., or about 1200 to about 200 I.U., preferably about 1600 I.U., alternatively about 10 to about 100 μg (microgram or mcg) of vitamin D, or about 10 to about 80 μg, or about 20 to about 60 μg, or about 30 to about 50 μg, preferably about 40 μg; alternatively the vitamin D can be present in about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μg of vitamin D;

3) vitamin C (L-ascorbic acid) or a salt thereof in ranges of about 10 to about 1000 mg, with a usual range of about 50 to about 500 mg and typically about 90 to about 200 or about 400 mg, or about 10 to about 250 mg, or about 20 to about 200 mg, or about 25 to about 150 mg, or about 30 to about 90 mg, or about 45 to about 75 mg, preferably about 60 mg; or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mg of vitamin C;

4) selenium (preferably as L-selenomethionine) in ranges of about 10 to about 250 μg, or about 20 to about 110 μg, or about 25 to about 100 μg, or about 27.5 to about 75 μg, or about 27.5 to about 55 μg, or about 40 to about 60, or about 41 to about 69 μg, preferably about 55 μg; or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 μg of L-selenomethionine;

5) zinc (preferably as zinc gluconate) in amounts of about 5 to about 100 mg, or about 5 to about 35 mg, or about 5 to about 20 mg, or about 6 to about 12 mg, or about 8 to about 14 mg, preferably about 11 mg; or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg of zinc gluconate;

6) folate (preferably as L-5-methyltetrahydrofolate) in amounts of about 0.1-1 mg (about 100 to about 1000 μg), or about 250 to about 800 μg, or about 300 to about 600 μg, or about 375 to about 625 μg, or about 400 to about 550 μg, preferably about 500 μg; or about 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 μg of L-5-methyltetrahydrofolate;

7) lycopene in amounts of about 0.5 to about 20 mg, or about 3 to about 12 mg, with usual ranges of about 1 to about 10 mg, or about 5 to 2 about 0 mg, or about 5 to about 15 mg, or about 7.5 to about 12.5 mg, preferably about 10 mg; or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.6, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg of lycopene;

8) a carnitine source (preferably as a blend of acetyl-L-carnitine HCl and propionyl-L-carnitine HCl) in amounts of about 1 to about 5 grams, with a usual range of about 2 to about 3 grams, or about 250 to about 4000 mg, or about 50 to about 1000 mg, or about 75 to about 800 mg, or about 200 to about 600 mg, or about 300 to about 500 mg, preferably about 400 mg; or about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, or 600 mg;

9) optionally, Coenzyme Q10 (CoQ10) up to about 100 mg, or up to about 75 mg, or up to about 50 mg, or up to about 30 mg, or up to about 25 mg, or up to about 20 mg, or about 10 to about 100 mg, or about 15 to about 75 mg, or about 20 to about 50 mg, or about 10 to about 30 mg, or about 15 to about 25 mg, preferably about 20 mg; or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg; and

10) optionally, glutathione in amounts of about 100 to about 1000 mg, with a usual range of about 400 to about 600 mg, preferably administered parenterally.

In another aspect of this invention, the combination is supplemented with alpha-lipoic acid in amounts of about 50 to about 600 mg.

Weight ratios (in mg) and relative ratios of the various ingredients for F1/6, F1/3 and FMAX are presented in the table below.

F1/6 F1/3 FMAX relative wt ratio relative wt ratio relative ratio of ingredients wt ratio (mg) ratio (mg) ratio (mg) ratio vit E:vit C 38:30 1.27:1 150:60 2.5:1 235:90 2.6:1 vit E:5MTHF 38:0.3 127:1 150:0.5 300:1 235:0.6 392:1 vit E:Zn 38:6 6.3:1 150:11 13.6:1 235:11 21.4:1 vit E:Se 38:0.0275 1382:1 150:0.055 2727:1 235:0.055 4273:1 vit E:carnitine 38:75 0.51:1 150:400 0.375:1 235:800 0.294:1 vit E:vit D 38:0.02 1900:1 150:0.04 3750:1 235:0.06 3917:1 vit E:lycopene 38:5 7.6:1 150:10 15:1 235:15 15.7:1 vit E:CoQ10 38:0 — 150:20 7.5:1 235:30 7.8:1 5MTHF:vit C 0.3:30 0.01:1 0.5:60 0.0083:1 0.6:90 0.0067:1 5MTHF:Zn 0.3:6 0.05:1 0.5:11 0.045:1 0.6:11 0.054:1 5MTHF:Se 0.3:0.275 10.9:1 0.5:0.055 9.09:1 0.6:0.055 10.9:1 5MTHF:carnitine 0.3:75 0.004:1 0.5:400 0.00125:1 0.6:800 0.00075:1 5MTHF:vit D 0.3:0.02 15:1 0.5:0.04 12.5:1 0.6:0.06 10:1 5MTHF:lycopene 0.3:5 0.06:1 0.5:10 0.05:1 0.6:15 0.04:1 5MTHF:CoQ10 0.3:0 — 0.5:20 0.025:1 0.6:30 0.02:1 carnitine:vit C 75:30 2.5:1 400:60 6.67:1 800:90 8.89:1 carnitine:Zn 75:6 12.5:1 400:11 36.4:1 800:11 72.7:1 carnitine:Se 75:0.0275 2727:1 400:0.055 7273:1 800:0.055 14545:1 carnitine:vit D 75:0.02 3750:1 400:0.04 10000:1 800:0.06 13333:1 carnitine:lycopene 75:5 15:1 400:10 40:1 800:15 53.3:1 carnitine:CoQ10 75:0 — 400:20 20:1 800:30 26.7:1 Zn:vit C 6:30 0.2:1 11:60 0.183:1 11:90 0.12:1 Zn:Se 6:0.0275 218:1 11:0.055 200:1 11:0.055 200:1 Zn:vit D 6:0.02 300:1 11:0.04 275:1 11:0.06 183:1 Zn:lycopene 6:5 1.2:1 11:10 1.1:1 11:15 0.73:1 Zn:CoQ10 6:0 — 11:20 0.55:1 11:30 0.37:1 Se:vit C 0.0275:30 0.00092:1 0.055:60 0.0092:1 0.055:90 0.0061:1 Se:vit D 0.0275:0.02 1.375:1 0.055:0.04 1.375:1 0.055:0.06 0.92:1 Se:lycopene 0.0275:5 0.0055:1 0.055:10 0.0055:1 0.055:15 0.0037:1 Se:CoQ10 0.0275:0 — 0.055:20 0.00275:1 0.055:30 0.0018:1 vit D:vit C 0.02:30 0.0067:1 0.04:60 0.00067:1 0.06:90 0.00067:1 vit D:lycopene 0.02:5 0.004:1 0.04:10 0.004:1 0.06:15 0.004:1 vit D:CoQ10 0.02:0 — 0.04:20 0.002:1 0.06:30 0.002:1 lycopene:vit C 5:30 0.17:1 10:60 0.17:1 15:90 0.17:1 lycopene:CoQ10 5:0 — 10:20 0.5:1 15:30 0.5:1 CoQ10:vit C 0:30 — 20:60 0.33:1 30:90 0.33:1

As is clear from this table, the relative weight ratio of:

natural vitamin E to vitamin C is about 1.2:1 to 2.7:1, preferably about 2.5:1;

natural vitamin E to L-5-methyltetrahydrofolate is about 120:1 to 400:1, preferably about 300:1;

natural vitamin E to zinc gluconate is about 6:1 to 22:1, preferably about 13.5:1, more preferably about 13.6:1;

natural vitamin E to L-selenomethionine is about 1300:1 to 4500:1, preferably about 2700:1, more preferably about 2727:1;

natural vitamin E to the acetyl- and propionyl-L-carnitine.HCl blend is about 0.25:1 to 0.55:1, preferably about 0.4:1, more preferably about 0.375:1;

natural vitamin E to vitamin D is about 1500:1 to 4000:1, more preferably about 3750:1;

natural vitamin E to lycopene is about 7:1 to 16:1, preferably about 15:1;

natural vitamin E to coenzyme Q10 is about 7:1 to 8:1, preferably about 7.5:1; L-5-methyltetrahydrofolate to vitamin C is about 0.006:1 to 0.015:1, preferably about 0.008:1, more preferably about 0.0083:1;

L-5-methyltetrahydrofolate to zinc gluconate is about 0.04:1 to 0.06:1, preferably about 0.045:1;

L-5-methyltetrahydrofolate to L-selenomethionine is about 9:1 to 11:1, preferably about 9.1:1, more preferably about 9.09:1;

L-5-methyltetrahydrofolate to the acetyl- and propionyl-L-carnitine.HCl blend is about 0.0007:1 to 0.005:1, preferably about 0.001:1, more preferably about 0.00125:1;

L-5-methyltetrahydrofolate to vitamin D is about 9:1 to 16:1, preferably about 12:1, more preferably about 12.5:1;

L-5-methyltetrahydrofolate to lycopene is about 0.03:1 to 0.07:1, preferably about 0.05:1;

L-5-methyltetrahydrofolate to CoQ10 is about 0.01:1 to 0.03:1, preferably about 0.02:1, more preferably about 0.025:1

Carnitine blend to vitamin C is about 2:1 to about 10:1, preferably about 6.7:1;

Carnitine blend to zinc gluconate is about 10:1 to about 80:1, preferably about 36:1;

Carnitine blend to L-selenomethionine is about 2500:1 to about 1500:1, preferably about 7273:1;

Carnitine blend to vitamin D is about 3500:1 to about 14000:1, preferably about 10000:1;

Carnitine blend to lycopene is about 10:1 to about 60:1, preferably about 40:1;

Carnitine blend to CoQ10 is about 15:1 to about 30:1, preferably about 20:1.

The relative ratio ranges of the other various ingredient combinations can be determined from the table above, with the preferred relative ratio being that of the F1/3 formulation.

Note that in all three F formulations the vitamin D:vitamin C ratio is maintained at 0.0067:1; the vitamin D:lycopene ratio is maintained at 0.004:1; the vitamin D:CoQ10 ratio is maintained at 0.002:1; the lycopene:vitamin C ratio is maintained at 0.17:1; the lycopene:CoQ10 ratio is maintained at 0.5:1; and the CoQ10:vitamin C ratio is maintained at 0.33:1.

In another aspect of the present invention, the combination therapy can optionally be supplemented with an aromatase inhibitor. Preferred aromatase inhibitor agents include natural and synthetic compounds that are able to modulate and reduce the activity of Aromatase, an enzyme found in the liver, which is responsible for the conversion of the androgens such as androstendione and testosterone into the estrogens such as estrone and estradiol. By inhibiting aromatase the body produces less estrogen and maintains a higher testosterone state. Suitable aromatase inhibitors include natural flavanoids such as chrysin, apigenin, indole-3-carbinols or synthetic compounds including but not limited to aminoglutethimide, anastrozole or 6-methylenandrosta-1,4-diene-3,17-dione.

In another embodiment, the antioxidant agent administered to the subject is a combination of the following antioxidant agents: vitamin C, vitamin E, selenium, zinc, and propionyl-L-carnitine. In yet another embodiment, the combination of the antioxidant agents consists essentially of vitamin C, vitamin E, selenium, zinc, propionyl-L-carnitine and folic acid. In yet another embodiment, the combination of the antioxidant agents consists of vitamin C, vitamin E, selenium, zinc, propionyl-L-carnitine and lycopene. In yet another embodiment, the combination of the antioxidant agents consists essentially of vitamin C, vitamin E, selenium, zinc, L-carnitine, folic acid and lycopene. In yet another embodiment, the combination of the antioxidant agents consists of vitamin C, vitamin E, selenium, zinc, acetyl-L-carnitine, folic acid and lycopene, and suitable excipients. In the most preferred embodiment, the combination of the antioxidant agents consists of vitamin C, vitamin E, selenium, zinc, acetyl-L-carnitine, proionyl-L-carnitine, folate or folic acid and lycopene and suitable excipients.

A suitable formulation (referred to herein as an F formulation) contains vitamin E (d-alpha-tocopheryl acetate) or, preferably, natural vitamin E, vitamin D (cholecalciferol), vitamin C (L-ascorbic acid or a salt thereof), selenium, zinc, folate or folic acid, lycopene, propionyl-L-carnitine and acetyl-L-carnitine, and optionally, Coenzyme Q10.

At least one embodiment of such formulation essentially contains natural vitamin E or d-alpha-tocopheryl acetate 200 I.U.; vitamin C (L-ascorbic acid or a salt thereof) 90-200 mg, selenium 55 μg, zinc 15 mg, folate or folic acid 0.5 mg, lycopene 5 mg, propionyl-L-carnitine 500 mg, and acetyl-L-carnitine 1 gram. Another embodiment contains vitamin C (L-ascorbic acid or a salt thereof) in amount of 90 mg. Yet in another embodiment, the formulation further contains lipoic acid in amount of 60 mg.

One formulation of the invention, sometimes referred to herein as F1/6, contains 30 mg of vitamin C, 300 μg of folate as L-5-methyltetrahydrofolate, 6 mg of zinc gluconate, 27.5 μg of L-selenomethionine, 75 mg of a blend of acetyl-L-carnitine and propionyl-L-carnitine HCl salts, 20 μg of vitamin D, 38 mg of natural vitamin E, and 5 mg of lycopene.

Another formulation of the invention, sometimes referred to herein as F1/3, contains 60 mg of vitamin C, 500 μg of folate as L-5-methyltetrahydrofolate, 11 mg of zinc gluconate, 55 μg of L-selenomethionine, 400 mg of a blend of acetyl-L-carnitine and propionyl-L-carnitine HCl salts, 40 μg of vitamin D, 150 mg of natural vitamin E, 10 mg of lycopene, and 20 mg of Coenzyme Q10.

Another formulation of the invention, sometimes referred to herein as FMAX, contains 90 mg of vitamin C, 600 μg of folate as L-5-methyltetrahydrofolate, 11 mg of zinc gluconate, 55 μg of L-selenomethionine, 800 mg of a blend of acetyl-L-carnitine and propionyl-L-carnitine HCl salts, 60 μg of vitamin D, 235 mg of natural vitamin E, 15 mg of lycopene, and 30 mg of Coenzyme Q10.

These formulations are presented in the following table, together with the dose ranges for the various ingredients considered to be safe and efficacious for the present purposes of treating male infertility or enhancing male fertility:

Safe and efficacious Ingredient F1/6 F1/3 FMAX range Vitamin C 30 mg 60 mg 90 mg about 10-250 mg Folate as L-5- 300 μg 500 μg 600 μg about 250-800 μg methyltetrahydrofolate Zinc gluconate 6 mg 11 mg 11 mg about 5-20 mg Selenium as L- 27.5 μg 55 μg 55 μg about 25-110 μg selenomethionine Acetyl- and propionyl- 75 mg 400 mg 800 mg about 50-1000 mg L-carnitine-HCl blend Vitamin D 20 μg 40 μg 60 μg about 10-80 μg (800 IU) (1600 IU) (2400 IU) (about 400-3200 IU) Natural Vitamin E 38 mg 150 mg 235 mg about 25-400 mg Lycopene 5 mg 10 mg 15 mg about 5-20 mg Coenzyme Q10 0 20 mg 30 mg about 20-50 mg

Thus, a composition for treating male infertility or enhancing male fertility, comprises vitamin C, a folate, a pharmaceutically acceptable zinc salt, a pharmaceutically acceptable selenium source, a carnitine blend comprising acetyl-L-carnitine and propionyl-L-carnitine or pharmaceutically acceptable forms thereof, vitamin D, natural vitamin E, lycopene, and coenzyme Q10. Preferably, the folate comprises L-5-methyltetrahydrofolate, the zinc salt comprises zinc gluconate, the selenium source comprises L-selenomethionine, the carnitine blend comprises acetyl- and propionyl-L-carnitine.HCL, and the natural vitamin E is the full mixture of naturally occurring vitamin E isoforms: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol. Preferably the vitamin C is present in about 30 to about 90 mg, the L-5-methyltetrahydrofolate is present in about 300 to about 600 μg, the zinc gluconate is present in about 6 to about 11 mg, the L-selenomethionine is present in about 27.7 to about 55 μg, the carnitine blend is present in about 75 to about 800 mg and comprises a blend of acetyl-L-carnitine.HCL and propionyl-L-carnitine.HCL, vitamin D is present in about 20 to about 60 μg, natural vitamin E is present in about 38 to about 235 mg, lycopene is present in about 5 to about 15 mg, and coenzyme Q10 is present in up to about 30 mg. More preferably the vitamin C is present in about 60 mg, the L-5-methyltetrahydrofolate is present in about 500 μg, the zinc gluconate is present in about 11 mg, the L-selenomethionine is present in about 55 μg, the carnitine blend is present in about 400 mg and is a blend of acetyl-L-carnitine.HCL and propionyl-L-carnitine.HCL, vitamin D is present in about 40 μg, natural vitamin E is present in about 150 mg, lycopene is present in about 10 mg, and coenzyme Q10 is present in about 20 mg. Preferably the vitamin C, the folate, the zinc salt, the selenium source and the carnitine blend are supplied together in a capsule; and the vitamin D, natural vitamin E, lycopene and coenzyme Q10 are supplied together in a softgel.

The amounts and relative ratios of the components of the composition are selected to effectively overcome the effects of oxidative stress on sperm as evidenced by reducing/controlling % DNA damage to less than or equal to 20%, and/or reducing/controlling % chromatin defects to less than or equal to 10%. In other embodiments the % DNA Damage is reduced/controlled to less than 10% or 15% or 20% or 25% or 30%. In still other embodiments the % chromatin defects are reduced/controlled to less than 5% or 10% or 20% or 25%.

The amounts and relative ratios of the components of the composition are selected to effectively overcome the effects of oxidative stress on sperm in vivo as indicated by restoration of pregnancy rate and/or survival rate.

A further aspect of the invention is directed to a kit for treating male infertility or enhancing male fertility, comprising:

-   -   a) a composition comprising:     -   vitamin C,     -   a folate,     -   a pharmaceutically acceptable zinc salt,     -   a pharmaceutically acceptable selenium source,     -   a carnitine blend comprising acetyl-L-camitine and         propionyl-L-camitine, or pharmaceutically acceptable forms         thereof,     -   vitamin D,     -   natural vitamin E,     -   lycopene, and     -   coenzyme Q10;         and     -   b) educational information or directions for use providing for         at least daily administration of said composition to a subject         in need thereof for about 6 weeks to about 12 months, until at         least one index for measuring sperm DNA damage is reduced         post-treatment by at least 2.5% as compared to the pretreatment         levels of the same index in said subject.

In one embodiment, the duration for the treatment is at least for six (6) weeks to about twelve (12) months. In another embodiment, the duration of the therapy is for three (3) months prior any fertilization attempts. In further embodiments, the duration of the treatment regime is for six (6) months prior any fertilization attempts. In this regard, a suitable therapy for either of the following formulations is two administrations per day of one of the combinations, formulations, or compositions described above for a period of at least three (3) months prior to any fertilization attempts. However, it will be appreciated that the administration of the disclosed formulation and the other agents in the various embodiments of the present invention may be within any time and frequency suitable to produce the desired effect.

The antioxidant and the other agents of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. Regardless of the route of administration selected, the combination of the ingredients are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

The amount of the active antioxidants which will be combined with a carrier material to produce a single dosage form will generally be that amount of the active ingredient(s) which is the optimal dose effective to produce the therapeutic effect, herein as measured by DFI. Methods of preparing pharmaceutical formulations or compositions include the step of bringing the antioxidants and/or other suitable active ingredients into association with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient(s) into association with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, microparticles, nanoparticles or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of the active ingredient(s).

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, powders, granules and the like), the prodrug(s), antioxidant(s) and/or other suitable pharmaceutically active ingredient(s) (in their micronized, microparticle or nanoparticle forms) is/are mixed with one or more pharmaceutically-acceptable carriers, such as cellulose, magnesium stearate, silicon dioxide, sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, cellulose and/or silicic acid; (2) binders, such as, for example, carboxymethyl-cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, silicon dioxide, other types of silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.

In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as cellulose, magnesium stearate, silicon dioxide, lactose or milk sugars, with or without high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient(s) moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow, extended or controlled release of the desired antioxident(s) therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, nanoparticles and/or microspheres. They may be sterilized by for example, filtration through a bacteria-retaining filter.

Liquid dosage forms for oral administration of the active ingredient(s) include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient(s), the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylacetate, butyl alcohol, benzyl benzoate, propylene glycol, glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, amyl alcohol, tetrahydrofuryl polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active ingredient(s), may contain suspending agents as, for example, ethoxylated alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, bentonite, agar and tragacanth, and mixtures thereof.

Dosage forms for the topical or transdermal administration of the active ingredient(s) include powders sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient(s) may be mixed under sterile conditions with pharmaceutically-acceptable carrier, and with any buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to the active ingredient(s), excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the active ingredient(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays and inhalers can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Compositions of the present invention may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. A transdermal delivery system provides for continuous administration throughout the treatment regimen. Transdermal patches have the added advantage of providing controlled delivery of the active ingredient(s) to the body. Such dosage forms can be made by dissolving, dispersing or otherwise incorporating the active ingredient(s) in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the active ingredient(s) across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the active ingredient(s) in a polymer matrix or gel.

Another mode of delivery for present combination of the present invention may be delivery via the use of targeting compounds. Accordingly a nanoparticulate or microparticulate blend of the antioxidant(s) in proper ratios may be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, polylactic acid, polyglycolic acid, copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels. Pharmaceutical compositions of this invention suitable for parenteral administration comprise the antioxidant(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain the combination of the antioxidants, minerals and vitamins, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, dextrose, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size, and by the use of surfactants. These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Injectable depot forms are made by forming microencapsule matrices of the antioxidant(s), minerals and vitamins in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the active ingredient(s) to polymer, and the nature of the particular polymer employed, the rate of release of the active ingredient(s) can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

The pharmaceutical compositions of the present invention may also be used in the form of veterinary formulations for treating infertile cattle, herd, or other infertile mammals, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or nonaqueous solutions or suspensions), tablets, boluses, powders, granules or pellets for admixture with feed stuffs, pastes for application to the tongue; (2) parenteral administration, for “ampule,” by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension or, when appropriate, by injection where a suspension or solution is introduced to the animal; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or any other methods fit to by those of ordinary skill in the art for administration to a region of interest.

Formulation of the agents of the present invention and their administration may be facilitated by an easy to use kit to ameliorate and assist patients to follow the treatment regimen. As discussed previously herein, the agents in the various embodiments of the present invention may be administered jointly or separately in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions).

Compositions containing the antioxidant agent and the other agents separately or jointly in the various embodiments of the present invention may also contain a preservative, stabilizer, dispersing agent, pH controller or isotonic agent Examples of suitable preservatives in the various embodiments of the present invention are glycerin, propylene glycol, phenol or benzyl alcohol. Examples of suitable stabilizers in the various embodiments of the present invention are dextran, gelatin, or alpha-thioglycerin.

Examples of suitable dispersing agents in the various embodiments of the present invention include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydrogenated castor oil 60. Examples of suitable pH controllers in the various embodiments of the present invention include hydrochloric acid, sodium hydroxide and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D-mannitol.

As discussed previously herein, the present invention is also suitable for reducing the generation of ROS in the male reproductive tract and/or semen. In this regard, the male reproductive tract will be understood to include the epididymis, the penis, the prostate gland, the seminal vesicles, the testes, the vas deferens and semen. Methods for determining the level of free radicals in the male reproductive tract are known in the art. For example, free radical production by sperm or seminal leukocytes can be measured directly using chemiluminescence assays known in the art. Alternatively, assays that measure free radical related damage to sperm lipid membrane may be used. For example, the TBARs assay (which involves the measurement of malondialdehyde, a marker of sperm membrane oxidation) or LPO-586 spectrophotometric assay may be used. These methods are described in Gomez et al (1998) International Journal of Andrology 21(2):81-96.

In one embodiment, the level of male fertility track is assessed by the way of suitable inflammatory indices. For example, one of ordinary skill in the art would with to assess the presence or absence of inflammatory cytokine, like one or more of IL-I, IL-6, IL-, TNF-α and Interferon-γ. Methods for determining the level of an inflammatory cytokine in the male reproductive tract are known in the art, such ELISA assays for detection of pro-inflammatory cytokines such as IL-I, IL-6, IL-8, TNF-α and Interferon-γ, as described in Depuydt et al. J Andrology 17(61), 699-707 (1996) or Maegawa et al J Reprod Immunology, 54, 33-42 (2002), and Nallella et al. Urology 64(5), 1010-3 (2004).

Those of ordinary skill in the art can also employ other methods of assessing the “sperm function.” The term as used herein includes any key component of sperm physiology and includes swimming activity towards the oocyte (motility), ability to undergo capacitation to penetrate the oocyte's outer coat (zona pellucida) and fuse with the oocyte membrane, and maintenance of sperm DNA integrity to form a functional male pro-nucleus at syngamy. Methods for determining the level of sperm function are known in the art. For example, suitable methods are described in detail within the World Health Organization (WHO) laboratory manual for the examination of human semen and sperm-cervical mucous interaction. 4th edition. Cambridge University Press 1999.

In at least one embodiment of the present invention, the sperm DNA fragmentation is assessed by SCSA. Evenson D P, et al, Relationship Between Assisted Reproductive Techniques (ART) outcome and Status of Chromatin Integrity as Measured by the Sperm Chromatin Structure Assay (SCSA). Hum. Reprod., 15, 1717-1722 (2000). At least one indicator of the clinical benefits of the claimed methodology is marked improvement of the DFI.

In principle, spermatozoa chromatin is different in nature from somatic cells chromatin. This difference is due to ploidy and DNA packaging, which is influenced by the replacement of histones in spermatocytes by transition proteins and then substitution of histone by protamine in spermatides. Green et al, Synthesis and Processing of Mammalian Protamines and Transition Proteins. Mol. Repr. Dev. 37, 255-263 (1994). Histones are the chief protein components of chromatin that coils around DNA enabling it to fit the large genomic information inside cell nuclei. Without histones, the unwound DNA in chromosomes would be very long. Replacement of histones by protamine are generally known to contribute to sperm's head condensation and DNA stabilization. See Id. Those of ordinary skill in the art can appreciate that DNA fragmentation levels above 30% as measured by SCSA are not compatible with initiation and maintenance of a term pregnancy. See Larson supra. SCSA test values, expressed as DFI, are significantly correlated with pregnancy rate in vivo and in vitro. In studies that included more than 25 couples undergoing in vitro fertilization and intracytoplasmic sperm injection cycles, no term pregnancy occurred when the DFI measurement was more than 27% in the semen samples utilized in these cycles. See Larson supra.

In the preferred embodiment of the present invention, baseline DFI is measured prior to the administration of the antioxidant regimen to assess a fertility capacity of male subjects. In the present invention, DFI values in combination with other subjective or objective signs of infertility would be used as the criteria to determine a couple's predisposition to failed pregnancy. Once study couples have undergone the baseline evaluation, they will receive a three (3) months course of therapy of the claimed antioxidant formulation in a kit containing other educational material regarding their therapy.

The effectiveness of the treatment course is then substantiated by statistically significant reduction in DFI and further the actual pregnancy of the couple's female subject. Those of ordinary skill in the art can appreciate measurements of other sperm parameters during the period of the trial to assess treatment efficacy. Such parameters can include sperm count, sperm density, motility, membrane integrity, testosterone or other hormonal levels if so desired, and other DNA damage techniques at various intervals including the entry period, 6 weeks, and/or 12 week time points. However, the actual clinical end point can be evaluated in view of other indicators including but not limited to fertilization rates among treatment group, embryo or blastocyst quality, number of viable embryos or blastocyst for transfer, number of miscarriages and the occurrence rate thereof.

An effective amount of the antioxidant agents and the other agents may be appropriately chosen, depending upon, for example, the type and extent of reduced fertility to be treated, the age and body weight of the subject, the DFI measurement, the frequency of administration, and the presence of other active agents. The instant antioxidant, mineral and vitamin combination, and/or the anti-aromatase agent may be administered to the subject separately or in combination via separate routes of administrations.

Accordingly, in another embodiment the present invention provides a combination product for improving sperm function in a male subject, the combination kit product including the following components: (a) a composition containing the present antioxidant, mineral and vitamin combination; and/or (b) an aromatase inhibitor agent, and/or (c) another adjuvant agent such as gluthothione or Coenzyme Q10 with (d) educational information and direction of use.

In yet another embodiment, the use of a carnitine source in the inventive combinations is described. L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine are endogenous ligands that play an essential role in transporting fatty acids into the mitochondria, where they are oxidized to produce energy. The carnitine pool is particularly important to spermatozoa as they are rich in mitochondria which generate energy especially to support their motility. Carnitines are also scavengers of oxygen free radicals in mammalian tissues. Thus, the biochemical and antioxidant role of carnitines make them vitally important molecules in the overall health of the spermatozoa.

Propionyl-L-carnitine is known in the art to increase cellular levels of L-carnitine by binding to the enzyme carnitine acetyltransferase (CAT) which is subsequently converted into propionyl-coenzyme A and free carnitine. Propionyl-L-carnitine is also rapidly absorbed after oral administration, reaching peak concentrations in about 1 hour followed by a rise in L-carnitine plasma levels 2-6 hours after the administration.

WO publication 03/084526 has previously shown the impact of such prodrugs in sperm motility and concentration. However, the acetyl-L-carnitine and propionyl-L-carnitine alone or in combination have not been described in male prenatal compositions. In at least one aspect of the present invention, it is believed that the exogenous propionyl-L-carnitine supplementation alone will more effectively address any carnitine deficiency in infertile men.

The forgoing examples are used to further describe the invention employing at least one embodiment of the presently disclosed combination, disclosed herein as the F profertility therapy. Those of ordinary skill in the art can appreciate that patients who had received IVF before and during pregnancy, as well as those patients resorting to other fertilization attempts, would benefit from the presently disclosed treatment regimen by improving their embryonic quality and reducing the complications during the pregnancy term. At least one embodiment of the present invention focuses the treatment regimen on the male partners. Yet another embodiment of the invention includes independently or simultaneously administering the regimen to the female partners.

With well over 3 million men currently are experiencing male related infertility in the United States. Traditionally male infertility treatment has not endeavored to ameliorate the underlying causes of infertility but rather used “mechanical” techniques such as intra-uterine insemination or IVF-ICSI to bypass the defect in sperm function. While these two techniques are undeniably successful in a large proportion of patients, they simply do not work or have very limited efficiency in other couples. It is likely that in many cases, sperm DNA fragmentation is responsible for the poor pregnancy outcome despite ART treatment. The present invention provides treatments that can prophylactically treat sperm DNA fragmentation and is likely to boost both natural and ART related pregnancy rates.

A total of two separate human trials are envisioned for further assessing the efficacy of the combination therapy instantly disclosed. Prior experiences with antioxidant combinations have not provided any evidence that antioxidant combinations can reduce sperm's DNA fragmentation secondary to oxidative stress. See Tremelton supra. Contrary to such a body of scientific evidence, the present treatment regimen unexpectedly show a significant reduction of DFI after three (3) months of therapy.

In assessing the efficacy of other prior art antioxidant combinations, one of ordinary skill in the art would appreciate that even though the rate of pregnancy outcomes might have improved in the antioxidant treatment groups, the measured sperm parameters for the treatment group were not affected and the treatment had no effect on sperm concentration, motility or morphology. See Tremelton supra at pages 219-220. Furthermore, the LPO-586 assay for lipid peroxidation damage did not detect any significant difference in levels of free radical damage to the sperm membrane of the subjects. See Id. In fact, prior art formulations failed to show any correlation between SOS or sperm DNA damage and pregnancy rates, since the sperm DNA damage assays employed failed to show a difference between the treatment and the placebo groups. See Id.

The present invention provides a new combination of antioxidants with an unexpected reduction in sperm's DNA fragmentation due to local oxidative stress. Unexpected results also include the observation of improved quality of the sperm parameters, which when coupled with successful pregnancies, confirms the efficacy of F formulation therapy in treating male infertility associated with SOS.

EXAMPLES Example 1

First combination for men is prepared according to the following formula:

TABLE 1 d-alpha-tocopheryl acetate 200 IU L-Ascorbic acid 200 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg Propionyl-L-Carnitine 0.5 g Excipients As needed

Example 2

A second combination is prepared according to the following formula:

TABLE 2 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg Acetyl-L-Carnitine 1.0 g Propionyl-L-Carnitine 0.5 g Excipients As needed

Example 3

Another formulation is prepared according to the following formula:

TABLE 3 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg L-Carnitine 1 g Excipients As needed

Example 4

Another formulation is prepared according to the following formula:

TABLE 4 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Acetyl-L-Carnitine 0.5 g Excipients As needed

Example 5

Another formulation is prepared according to the following formula:

TABLE 5 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Propionyl-L-Carnitine 0.5 g Excipients As needed

Example 6

Another formulation for men is prepared according to the following formula:

TABLE 6 Vitamin E 200 IU L-Ascorbic acid 90 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg Propionyl-L-Carnitine 0.5 g Excipients As needed

Example 7

Yet another formulation is prepared according to the following formula:

TABLE 7 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg Acetyl-L-Carnitine 0.75 g Propionyl-L-Carnitine 0.5 g Excipients As needed

Example 8

Another formulation is prepared according to the following formula:

TABLE 8 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg L-Carnitine 1 g Excipients As needed

Example 9

Another formulation is prepared according to the following formula:

TABLE 9 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Acetyl-L-Carnitine 0.5 g Excipients As needed

Example 10

Another formulation is prepared according to the following formula:

TABLE 10 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Propionyl-L-Carnitine 0.5 g Excipients As needed

Example 11

Couple DP underwent multiple cycles of IVF that resulted in no viable pregnancy. The sperm DNA fragmentation index of the male subject was measured by SCSA to be 27%. The male partner then started on a twice a day combination therapy described in Example 3, Table 3 for a period of three (3) months. The combination contained vitamin E (d-alpha-tocopheryl acetate) 200 I.U., vitamin C 200 mg, selenium 55 μg, zinc 15 mg, folic acid 0.1 mg, L-carnitine 1 g, and lycopene 5 mg.

The post treatment DFI measurement showed an absolute 7.4% reduction in the DFI levels amounting to a 27.4% improvement from the pretreatment baseline DFI levels. Upon completion of the therapeutic regimen, even though no in vitro embryo assessment was done, the female partner became pregnant and gave birth to a healthy child nine (9) months following the termination of the antioxidant therapy.

Example 12

In another trial, the same couple used the same treatment regimen as described in Example 2. The male's sperm DFI measurement indicated a free radical damage of 39.6% prior to the initiation of the antioxidant combination therapy. The DFI measurement at least three (3) months after the initial treatment was measured as 27.2 showing an unexpected improvement of at least an absolute 12.4% reduction in DNA fragmentation index amounting to a 31% improvement from the pretreatment baseline DFI levels. While no in vitro embryo assessment was done, the female partner became pregnant and gave birth to a healthy child nine (9) months thereafter.

While the invention has been described with references to specific embodiments, modifications and variations of the invention may be construed without departing from the scope of the invention, which is defined in the following claims.

Example 13

Mouse Sperm DNA Damage Assessment

Evaluation of Sperm DNA Integrity

Sperm DNA integrity was assessed by acridine orange (AO) staining. Initially, 20 μl of washed epididymal sperm were smeared on slides and fixed with Carnoy's solution (methanol/acetic acid, 3:1; Merck, Darmstadt, Germany) for two hours at −4° C. Then, slides were rinsed with PBS (phosphate-buffered saline) buffer and stained with acridine orange in citrate phosphate buffer (80 mL citric acid 0.1 M+5 mL NaH₂PO₄ 0.3 M, pH 2.5) for 90 min. Slides were then rinsed with PBS buffer and evaluated using a fluorescent microscope (100× magnification, Olympus BX51, Tokyo, Japan). Sperm with green dye in the head region were considered as sperm with normal DNA, while sperm with orange/red dye in the head region were considered as sperm with DNA damage. 200 spermatozoa on each slide were assessed and the results were expressed as a percentage of sperm DNA damage.

As shown in FIG. 1, the synthetic drugs efavirenz, acetaminophen and paroxetine all increase mouse sperm % DNA damage versus control. Of the three F formulations, F1/6, F1/3 and FMAX, surprisingly only the F1/3 formulation restores the control level of % DNA damage. The low dose of F1/3 is too low to reverse the damage to mouse sperm DNA, while the high dose of the FMAX formulation appears to be damaging due to reductive stress.

Example 14

Mouse Sperm Chromatin Defect Assessment

Evaluation of Sperm Chromatin Maturity

For assessment of presence of excessive histones by Aniline Blue (AB) staining or chromatin maturation, a smear for each sample was prepared by placing 20 μl of washed epididymal sperm on a glass slide which was subsequently air dried and fixed with 3% buffered glutaraldehyde in 0.2 M phosphate buffer (pH=7.2) for 2 h at room temperature. Then each smear was treated with 5% aqueous AB stain in 4% acetic acid for 5 min, and rinsed twice with PBS buffer. Finally, at least, 200 spermatozoa were randomly assessed using a light microscope (100× magnification, Olympus CX31, Japan). Unstained or pale blue-stained sperm were considered normal chromatin compaction or sperm with normal content of histones, while dark blue stained sperm were considered sperm with residual histones or abnormal chromatin compaction as aniline blue positive (AB+).

As shown in FIG. 2, the synthetic drugs efavirenz, acetaminophen and paroxetine all increase mouse sperm % chromatin defects versus control. Again, of the three F formulations, F1/6, F1/3 and FMAX, surprisingly only the F1/3 formulation restores the control level of % chromatin defect. The low dose of F1/3 is too low to reverse the damage to mouse sperm DNA, while the high dose of the FMAX formulation appears to be damaging due to reductive stress.

Example 15

Histology

For histological studies, testicular tissue samples were fixed in Bouin's solution and processed using the standard histological method. Paraffin blocks were sectioned at 5-6 μm and stained with Hematoxylin and Eosin (H&E).

Example 16

Mouse Pregnancy Outcomes, General Protocol

At 13 weeks of age (30 days after the start of gavaging), male mice of each group were divided into 6 groups and kept in separate cages. Sexually mature, 7-8-week-old female mice were mated with fertile males (female:male ratio, 2:1) for two days, and vaginal plugs were checked the next morning. Mating was confirmed by observing the presence of a vaginal plug, and the day of positive vaginal plug observation was considered to be Day 1 of pregnancy (D1). The females that did not show a plug were excluded from the study. This was repeated twice with 4 new female mice. Pregnant mice were also monitored daily for signs of abortion.

Mated female mice from each group were humanely euthanized using CO₂ on day 19 of pregnancy. The total number of fetuses were counted.

The litters of 2 female mice were kept, and their numbers were counted 2 and 30 days after birth to assess the survival rate in different groups.

Pregnancy Outcomes for Efavirenz, Acetaminophen and Paroxetine

As shown in FIG. 3, efavirenz strongly reduces the pregnancy rate and the survival rate of the litters. The F1/6 and F1/3 formulations restore the pregnancy rate, but there is a drop in pregnancy rate for the FMAX formulation. Also, with regard to the survival rate of the litters, only the F1/3 formulation completely restores pup survival.

As shown in FIG. 4, even as mild a drug as acetaminophen (ACE) strongly reduces the pregnancy rate and also reduces the survival rate of the litters. Only the F1/3 formulation restores the pregnancy rate. The survival rate of the pups is improved by all three F formulations, although the litter numbers are improved only by the F1/6 and F1/3 formulations.

As shown in FIG. 5, paroxetine strongly reduces the pregnancy rate. Again, the F1/3 formulation shows the best response in terms of restoring the pregnancy rate, and FMAX is least effective. Also, the litter numbers are superior to controls only for the F1/3 formulation, with the FMAX formulation having a poorer in vivo result.

These experiments demonstrate that: 1) environmental factors such as synthetic drugs, regardless of their chemical structures, or perceived mildness or harshness, can increase oxidative stress and impair semen quality measured by a variety of markers such as sperm concentrations or motility; this was shown for all three drugs, efavirenz, acetaminophen and paroxetine; and 2) surprisingly, there is an optimum dose of the presently claimed antioxidant/vitamin/mineral combination to block, reverse or overcome the oxidative stress effects on semen from the chronic use of such synthetic drugs.

As noted above, damage appears at three levels. FIGS. 1 and 2 show damage at the cellular level as indicated by biomarkers of cellular damage. Thus DNA damage/chromatin defects are reversed with the F1/3 dose, the lower F1/6 dose is ineffective, and the FMAX dose appears to be damaging via reductive stress. At the tissue level, cellular morphology and tissue histology of sections of the seminiferous tubules compared with controls show the same pattern of optimal response with the F1/3 dose (data not shown). Likewise, at the in vivo level, pregnancy outcomes show significant differences between live birth rates and viability between the three doses, with the F1/3 dose being optimal in terms of restoring the pregnancy and/or survival rates. See FIGS. 3, 4 and 5.

However, these are controlled studies involving a single drug that causes oxidative stress. In men in vivo, the sources of oxidants are multiple, from heat and radiation to numerous chemical xenobiotic entities. Identifying the sources of such a collective insult in men and estimating the effective dose of the present inventive combination to reverse their aggregate effects is not straightforward, but still can be qualitatively assessed by the skilled clinician. 

What is claimed is:
 1. A method of overcoming oxidative stress in a subject in need thereof, comprising at least daily administering to said subject a composition comprising: vitamin C, a folate, a pharmaceutically acceptable zinc salt, a pharmaceutically acceptable selenium source, a carnitine blend comprising acetyl-L-carnitine and propionyl-L-carnitine, or pharmaceutically acceptable forms thereof, natural vitamin E, and lycopene, wherein the duration of treatment is from about 6 weeks to about 12 months; and wherein the amounts of vitamin C, folate, zinc salt, selenium source, carnitine blend, natural vitamin E, and/or lycopene are selected to reduce/control % DNA damage to less than or equal to 20%, and/or reduce/control % chromatin defect to less than or equal to 10%.
 2. The method of claim 1, wherein the composition further comprises vitamin D and/or coenzyme Q10, wherein the amounts of vitamin C, folate, zinc salt, selenium source, carnitine blend, natural vitamin E, lycopene, vitamin D and/or coenzyme Q10 are selected to reduce/control % DNA damage to less than or equal to 20%, and/or reduce/control % chromatin defect to less than or equal to 10%.
 3. The method of claim 1, wherein the oxidative stress is manifested as male infertility or reduced male fertility in the subject.
 4. The method of claim 2, wherein the oxidative stress is manifested as male infertility or reduced male fertility in the subject.
 5. The method of claim 4, wherein the folate comprises L-5-methyltetrahydrofolate, the zinc salt comprises zinc gluconate, the selenium source comprises L-selenomethionine, and the carnitine blend comprises acetyl-L-carnitine.HCL and propionyl-L-carnitine.HCL.
 6. The method of claim 5, wherein vitamin C is present in about 30 to about 90 mg, L-5-methyltetrahydrofolate is present in about 300 to about 600 μg, zinc gluconate is present in about 6 to about 11 mg, L-selenomethionine is present in about 27.7 to about 55 μg, the carnitine blend is present in about 75 to about 800 mg, vitamin D is present in about 20 to about 60 μg, natural vitamin E is present in about 38 to about 235 mg, lycopene is present in about 5 to about 15 mg, and coenzyme Q10 is present in up to about 30 mg.
 7. The method of claim 5, wherein vitamin C is present in about 60 mg, L-5-methyltetrahydrofolate is present in about 500 μg, zinc gluconate is present in about 11 mg, L-selenomethionine is present in about 55 μg, the carnitine source is present in about 400 mg, vitamin D is present in about 40 μg, natural vitamin E is present in about 150 mg, lycopene is present in about 10 mg, and coenzyme Q10 is present in about 20 mg.
 8. The method of claim 1, wherein vitamin C, the folate, the zinc salt, the selenium source and the carnitine blend are supplied together in a capsule; and vitamin D, natural vitamin E, lycopene and coenzyme Q10 are supplied together in a softgel, wherein the capsule and the softgel are administered together.
 9. The method of claim 7, which is effective to overcome the effects of oxidative stress on sperm as indicated by % DNA damage.
 10. The method of claim 7, which is effective to overcome the effects of oxidative stress on sperm as indicated by % chromatin defect.
 11. The method of claim 9, which is effective to overcome infertility in vivo as indicated by restoration of pregnancy rate and/or survival rate of offspring.
 12. The method of claim 10, which is effective to overcome infertility in vivo as indicated by restoration of pregnancy rate and/or survival rate of offspring.
 13. The method of claim 6, wherein the relative weight ratio of natural vitamin E to L-5-methyltetrahydrofolate is about 120:1 to 400:1.
 14. The method of claim 6, wherein the relative weight ratio of natural vitamin E to L-5-methyltetrahydrofolate is about 300:1.
 15. The method of claim 6, wherein the relative weight ratio of natural vitamin E to the carnitine blend is about 0.25:1 to 0.55:1.
 16. The method of claim 6, wherein the relative weight ratio of natural vitamin E to the carnitine blend is about 0.375:1
 17. The method of claim 6, wherein the relative weight ratio of L-5-methyltetrahydrofolate to the carnitine blend is about 0.0007:1 to about 0.005:1.
 18. The method of claim 6, wherein the relative weight ratio of L-5-methyltetrahydrofolate to the carnitine blend is about 0.00125:1. 